Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B (NANB) viral hepatitis, infecting approximately 4 million people in the United States. HCV is an enveloped RNA virus containing a positive sense and single-stranded RNA genome. The molecular mechanism of HCV RNA replication is not well understood. Circumstantial evidence derived from biochemical and reverse genetics studies suggests that HCV RNA genome is likely replicated in a membrane-bound protein complex consisting of viral nonstructural proteins and probably also cellular proteins. The composition of the viral replication complex has yet to be determined. The overall goal of this research application is to determine viral and cellular components of the HCV replication complex using cell biological, immunochemical, and proteomic approaches. We have already constructed a hepatoma cell line (Huh7) that maintains a high level of replication of a subgenomic HCV RNA. High levels of viral nonstructural proteins have also been expressed in Huh7 cells transfected with a DNA vector and co-infected with a recombinant vaccinia virus expressing a T7 RNA polymerase. These cell culture systems will be used for isolation and identification of the viral replication complex. Intracellular localization of the replication complex will be determined by immunofluorescence confocal microscopy. The replication complex will be isolated by subcellular fractionation coupled with an in vitro assay for viral RNA replication. Both viral and cellular proteins of the replication complex will be determined by immunoprecipitation, immunoblot, two-dimensional gel electrophoresis, and proteomic mass spectrometry. Information gained from these studies will immediately contribute to our understanding of the molecular mechanism of HCV RNA replication. Furthermore, findings derived from studies detailed in this application will provide novel targets for discovery of specific and effective agents inhibiting HCV RNA replication.